Production of human translation-competent lysates using dual centrifugation

Author:

Gurzeler Lukas-AdrianORCID,Ziegelmüller Jana,Mühlemann OliverORCID,Karousis Evangelos D.ORCID

Abstract

ABSTRACTProtein synthesis is a central process in gene expression and the development of efficient in vitro translation systems has been the focus of scientific efforts for decades. The production of translation-competent lysates originating from human cells or tissues remains challenging, mainly due to the variability of cell lysis conditions. Here we present a robust and fast method based on dual centrifugation that allows for detergent-free cell lysis under controlled mechanical forces. We optimized the lysate preparation to yield cytoplasm-enriched extracts from human cells that efficiently translate mRNAs in a cap-dependent as well as in an IRES-mediated way. Reduction of the phosphorylation state of eIF2α using recombinant GADD34 and 2-aminopurine considerably boosts the protein output, reinforcing the potential of this method to produce recombinant proteins from human lysates.

Publisher

Cold Spring Harbor Laboratory

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